Pericellular Spaces Research Articles

A Histological Study of the Effects of Administration and Withdrawal of Anabolic Androgenic Steroid (Nandrolone Decanoate) on the Hippocampus and the Prefrontal Cortex in Adult Male Albino Rats

Abstract Background The PFC and the hippocampus are parts of the limbic systems and have a major role in emotion regulation, memory and learning. Anabolic androgenic steroids (AASs) are large groups of synthetic derivatives of testosterone, prescribed for the treatment of male hypogonadism. Common abuse of AASs has spread among the general population and Aim of the Work The aim of the current work was to study the effects of AASs on the PFC and the hippocampus histologically and statistically and to evaluate the reversibility of these effects in these regions after AASs withdrawal. Material and Methods 24 adult male albino rats (3 – 6) months old (weighing 200-225 gm) were used in the current study and divided into 3 groups; according to the procedure and treatment type: control group (I) (12 rats) and AASs treated group (II) (6 rats) and Withdrawal group (III) (6 rats). Fixation for the brain using bouin solution was done first in situ followed by its removal and transfer to a glass bottle containing bouin solution for 24 hours to ensure complete fixation. The PFC and the hippocampi were obtained through parasagittal section. Specimens were processed for preparation of paraffin blocks then stained with H&E stain and immunohistochemically stained for GFAP antibody. Image analysis &morphometry were performed on stained sections for; number of surviving pyramidal neurons and the mean area percentage of GFAP staining. Results On examination of H&E-stained sections of control groups, the PFC was formed of the six- layers, the molecular layer, outer granular layer, outer pyramidal layer, inner granular layer, inner pyramidal layer and the multiform layer. The Hippocampus was formed of two components: the dentate gyrus (DG), the hippocampus proper (Cornu Ammonis, with its four regions: CA1, CA2, CA3 and CA4). The cells forming the hippocampus proper were arranged in three layers; the molecular, the pyramidal and the polymorphic layers. In immunohistochemically stained section, the present work showed minimal apparent immune-reaction in the cytoplasm and processes of astrocytes in the PFC and CA1 region of the hippocampus in the control group. On examination of H&E-stained sections of the AASs treated groups, signs of neuronal damage were observed. The inner pyramidal layer of the PFC and the pyramidal layer of CA1 region of the hippocampus revealed abnormal shrunken pyramidal cells having deeply stained nuclei, peri-cellular spaces and flame like appearance with pointed end. Moreover, these layers demonstrated also normal pyramidal cells showing also large rounded open vesicular nuclei and prominent nucleoli. Immunohistochemically stained sections of the treated group in both regions showed apparent extensive brownish immunostaining of the astrocytes with ramifying processes along the layer. Examination of H&E-stained sections of the withdrawal group revealed the inner pyramidal layer of the PFC and the pyramidal layer of CA1 region of the hippocampus showing normal pyramidal cells having large rounded open vesicular nuclei, prominent nucleoli and apical dendrites. It also showed dark irregular cells having deeply stained nuclei, peri- cellular spaces and flame like appearance with pointed end. Immunohistochemically stained section of the treated group in both regions showed apparent minimal brownish immunostaining of the astrocytes. Conclusion Results of the present experimental study demonstrated that AASs (in the form of ND) administration affecting the PFC and the hippocampus resulted in several histopathological and morphometric harmful effects which regressed to a lesser extent through its withdrawal after 2 weeks.

Neuroprotective Assessment of Betaine against Copper Oxide Nanoparticle-Induced Neurotoxicity in the Brains of Albino Rats: A Histopathological, Neurochemical, and Molecular Investigation.

Copper oxide nanoparticles (CuO-NPs) are commonly used metal oxides. Betaine possesses antioxidant and neuroprotective activities. The current study aimed to investigate the neurotoxic effect of CuO-NPs on rats and the capability of betaine to mitigate neurotoxicity. Forty rats; 4 groups: group I a control, group II intraperitoneally CuO-NPs (0.5 mg/kg/day), group III orally betaine (250 mg/kg/day) and CuO-NPs, group IV orally betaine for 28 days. Rats were subjected to neurobehavioral assessments. Brain samples were processed for biochemical, molecular, histopathological, and immunohistochemical analyses. Behavioral performance of betaine demonstrated increasing locomotion and cognitive abilities. Group II exhibited significantly elevated malondialdehyde (MDA), overexpression of interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α). Significant decrease in glutathione (GSH), and downregulation of acetylcholine esterase (AChE), nuclear factor erythroid 2-like protein 2 (Nrf-2), and superoxide dismutase (SOD). Histopathological alterations; neuronal degeneration, pericellular spaces, and neuropillar vacuolation. Immunohistochemically, an intense immunoreactivity is observed against IL-1β and glial fibrillary acidic protein (GFAP). Betaine partially neuroprotected against CuO-NPs associated alterations. A significant decrease at MDA, downregulation of IL-1β, and TNF-α, a significant increase at GSH, and upregulation of AChE, Nrf-2, and SOD. Histopathological alterations partially ameliorated. Immunohistochemical intensity of IL-1β and GFAP reduced. It is concluded that betaine neuroprotected against most of CuO-NP neurotoxic effects through antioxidant and cell redox system stimulating efficacy.

Histopathological and ultrastructural changes in the rats brain after air shock wave impact

The using of explosive substances and devices can lead to brain injuries, the diagnosis of which requires the determination of primary biomarkers. Therefore, the aim was to establish and study histopathological and ultrastructural changes in the rats brain after exposure to an air blast wave. The study was carried out on 18 male Wistar rats, which were randomly divided into two groups: sham (n=9) and experimental (n=9). The animals of the experimental group were anesthetized with halothane and gently fixed in a horizontal position on the abdomen with the front part of the rat's muzzle at a distance of 5 cm from the device opening and subjected to an excess pressure of 26-36 kPa. Animals of both groups were decapitated, brains were removed, fixed, histopathological and ultrastructural analyzes were conducted using standard methods. Intergroup differences were assessed by Mann-Whitney U-test. Light microscopy revealed primary lesions in the form of small focal, multifocal hemorrhages, cerebral vessel ruptures and microscopic ruptures of the brain substance. In almost all brain samples, there is a significant saturation of the venous vessels with the presence of erythrocyte stasis. Violation of the blood-brain barrier, the presence of edema of the perivascular space, and petechial hemorrhages in the neuropil were registered ultrastructurally. Based on the study results, a morphological algorithm for assessing primary histostructural intracranial brain injuries and their consequences after exposure to an air shock wave was proposed. The injuries were found to be caused by the direct traumatic effect of the air shock wave. In the acute post-traumatic period, histopathological and ultrastructural changes in the brain can manifest as changes in neurons and in the blood-brain barrier and be accompanied by perivascular multifocal small-focal hemorrhages, neuropil ruptures, edema of pericellular and perivascular spaces, which together can be considered as biomarkers of primary traumatic changes after exposure to an air shock wave.

Effects of Aluminum Oxide Nanoparticles in the Cerebrum, Hippocampus, and Cerebellum of Male Wistar Rats and Potential Ameliorative Role of Melatonin.

Aluminum oxide nanoparticles (Al2O3 NPs) have been widely used in vaccine manufacture, food additives, human care products, and cosmetics. However, they also have adverse effects on different organs, including the liver, kidneys, and testes. Melatonin is a potent antioxidant, particularly against metals by forming melatonin-metal complexes. The present study aimed to investigate the protective effects of melatonin against Al2O3 NP-induced toxicity in the rat brain. Forty adult male Wistar rats were allocated to four groups: the untreated control (received standard diet and distilled water), Al2O3 NP-treated (received 30 mg/kg body weight Al2O3 NPs), melatonin and Al2O3 NP-treated (received 30 mg/kg body weight Al2O3 NPs + 10 mg/kg body weight melatonin), and melatonin-treated (received 10 mg/kg body weight melatonin) groups. All treatments were by oral gavages and administered daily for 28 days. Afterward, the rats were sacrificed, and samples from various brain regions (cerebrum, cerebellum, and hippocampus) were subjected to biochemical, histopathological, and immunohistochemical analyses. Al2O3 NPs substantially increased malondialdehyde, β-amyloid 1-42 peptide, acetylcholinesterase, and β-secretase-1 expression, whereas they markedly decreased glutathione levels. Furthermore, Al2O3 NPs induced severe histopathological alterations, including vacuolation of the neuropil, enlarged pericellular and perivascular spaces, vascular congestion, neuronal degeneration, and pyknosis. Al2O3 NP treatment also resulted in an intense positive caspase-3 immunostaining. Conversely, the administration of melatonin alleviated the adverse effects induced by Al2O3 NPs. Therefore, melatonin can diminish the neurotoxic effects induced by Al2O3 NPs.

Morphological aspects of diaphragm dysfunction in COVID-19-associated pneumonia

To assess morphological changes in the diaphragm and phrenic nerve in patients who died from COVID-19. In a case-control study, an analysis was made of autopsy material of the diaphragm and phrenic nerve of those who died from COVID-19 infection complicated by SARS-CoV-2-associated pneumonia, confirmed in vivo by the presence of SARS-CoV-2 RNA (Group 1, n=12), and those who died with a diagnosis of acute cerebrovascular accident of the ischemic type without parenchymal respiratory failure (Group 2, n=3). The main histopathological features in the diaphragm of the 1st group were the edema of the pericellular spaces of muscle fibers, edema of perivascular spaces, diapedese hemorrhages, plethora in arteriolas, in most veins and capillaries, red blood clots were revealed; in the diaphragmatic nerve - swelling of the perineral space, severe edema around the nerve fibers inside the nerve trunk. In the diaphragm of group 2, edema of pericellular spaces of muscle fibers and edema of perivascular spaces were less pronounced (p<0.001), hemorrhages were not determined; in the diaphragmatic nerve, moderate edema of the perineral space, mild swelling inside the nerve trunk around the nerve fibers was revealed (p<0.001). The glycogen content in the muscle cells of group 1 is significantly lower compared to group 2 (p<0.001). The study confirms the characteristic pathological picture of organ damage in COVID-19. However, the leading pathological mechanism of organ damage requires further investigation.

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy.

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

Spinning the Matrix – Mechanisms of Basement Membrane Secretion and Assembly

Basement membranes (BMs) are sheet‐like extracellular matrices that line the basal surfaces of epithelial tissues and surround muscles, adipose tissue, and nerves. My lab studies how regulated changes to BM architecture contribute to tissue morphogenesis. In Drosophila, elongation of the initially spherical egg chamber coincides with the generation of a polarized network of fibrils in its surrounding BM. We found that BM fibrils are assembled from newly synthesized proteins in the pericellular spaces between the egg chamber’s epithelial cells and undergo oriented insertion into the planar BM network by directed epithelial migration. We further found that a Rab10‐dependent secretion pathway ensures that a subset of new BM proteins exit the cell through a basal region of the lateral membrane for fibril formation. Rab10 recruits kinesin motors to BM secretory vesicles and transports them to this location on a polarized microtubule array. Finally, by manipulating Rab10 function, we showed for the first time that BM fibrils play an instructive role in egg chamber elongation. This work identifies a novel intracellular trafficking pathway for BM proteins and highlights how regulated protein secretion can synergize with tissue movement to build a polarized BM architecture that controls tissue shape.

The ameliorative effect of nanoselenium on histopathological and biochemical alterations induced by melamine toxicity on the brain of adult male albino rats

Melamine is a chemical substance used as a food adulterant because of its high nitrogen content; it is known to induce neurotoxicity, thereby adversely affecting the central nervous system. The biocompatibility, bioavailability, lower toxicity, and the large surface area of nanosized selenium relative to its other forms indicate that selenium nanoparticles (SeNPs) have a potential ameliorative effect against melamine-induced neurotoxicity. In this study, we tested this hypothesis using 40 adult male albino rats that were randomly assigned into four groups (n = 10 per group): group I rats served as the untreated negative controls and were fed with standard diet and distilled water; group II rats were orally treated with melamine (300 mg/kg body weight/d); group III rats orally received melamine (300 mg/kg body weight/d) and SeNPs (2 mg/kg body weight/d); and group IV rats received SeNPs only (2 mg/kg body weight/d) for 28 days. Blood and brain samples were collected from all rats and processed for biochemical, histopathological, and immunohistochemical investigations. SeNPs were encapsulated in starch as a natural stabilizer and a size-controlling agent (SeNP@starch). The prepared SeNPs were characterized using different techniques. Inductively coupled plasma-optical emission spectrometry (ICP-OES) indicated that the percentage of selenium loaded in starch was 1.888 %. Powder x-ray diffractometer (XRD) was used to investigate the crystalline structure of the Se-NP@starch, to be tubular and composed of amorphous starch as well as metallic selenium. Thermogravimetric analysis confirmed the thermal stability of the product and determined the interactions among the different components. Transmission electron microscope demonstrated the spherical shape of SeNPs and their dispersion into starch surface as well as evaluating their size in nanoscale (range 20−140 nm). Our results revealed that the melamine- exposed rats had significantly elevated in malondialdehyde levels, significantly reduced in total antioxidant capacity, down-regulated expression of the antioxidant related genes Nrf2 (nuclear factor erythroid 2–related factor 2) and GPx (glutathione peroxidase), as well as up-regulated expression of the apoptosis-related gene Bax (B-cell lymphoma 2–associated X protein), with down regulation of Bcl-2 (B-cell lymphoma 2). Histopathological examination exhibited several alterations in the cerebrum, cerebellum, and hippocampus of the treated rats compared with the controls. Neuronal degeneration, vacuolation of the neuropils, and pericellular and perivascular spaces were observed. In addition, the pyramidal and granular cell layers of the hippocampus and cerebellum, respectively, were found to have significantly reduced thickness. Furthermore, a significant decrease in the percentage area of the glial fibrillary acidic protein and a significant increase in the percentage area of caspase-3 were noted. On the other hand, co-treatment with SeNPs partially ameliorated these alterations. A significant reduction in malondialdehyde levels; a non- significant elevation in total antioxidant capacity; up-regulation, upregulation of Nrf2, GPx, and Bcl-2 and downregulation of Bax were recorded. Neuronal degeneration, vacuolation of neuropils, and pericellular spaces were reduced. The pyramidal and granular cell layers restored their normal thickness. The percentage area of the glial fibrillary acidic protein significantly increased, whereas that of caspase-3 significantly decreased. In conclusion, SeNPs have an ameliorative effect against melamine-induced neurotoxicity in albino rats.

Aluminium Induced Neurodegeneration in Rat Cerebrum in Presence of Ethanol Co-exposure

Introduction: Aluminium (AL) exposure leads to neurotoxicity and many problems in the body. AL role in Alzheimer's Disease (AD) is unknown and controversial to the scientists. According to World Health Organisation (WHO) provisional tolerable weekly intake of AL is 2 mg/kg body weight. Moderate intake of alcohol may favour body in coronary heart disease and diabetes mellitus, etc. Aluminium being cheaper along with increased consumption of alcohol, mixed with each other may induce neurotoxicity. Aim: The study was designed to identify the effects of AL in cerebrum of rats in presence of ethanol co-exposure. Materials and Methods: An experimental study was carried out at Dr. RP Government Medical College, Kangra and Government Medical College, Amritsar, India after due approval from the Institute Animal Ethics Committee. Thirty-two wistar rats were divided into one vehicle control and three experimental groups. Group I received the normal saline water as vehicle control group. Group II received AL chloride 4.2 mg/kg body weight as experimental group. Group III received ethanol 1 gm/kg body weight as experimental group. Group IV received both AL chloride 4.2 mg/kg body weight and ethanol 1 gm/kg body weight as experimental group. After treatment, brain cortex was processed for histopathological observation under microscope. Results: Cerebral cortex showed normal architecture of the brain with haematoxylin and eosin staining and cresyl violet staining and modified Bielchowsky silver staining in low and high magnification in vehicle control group. Experimental group treated with AL and ethanol separately showed reduction in the count of pyramidal cells with moderate neuronal degeneration with pyknotic nuclei. Vacuolar changes and pericellular spaces around the necrotic neurons were also seen. Combined AL and ethanol treated group showed acute neurodegeneration and necrosis of cortex indicating chromatolysis and loss of substances and Neurofibrillary Tangle (NFT) and plaque. Conclusion: It has been concluded that the ethanol induced the effects of AL on the cerebrum and plays a significant role in AD pathogenesis.

Protective effect of propolis on manganese chloride neurotoxicity of olfactory bulb in adult male albino rat.

Manganese (Mn) is widely used for industrial purposes and exposure to high levels of Mn may cause an irreversible brain disease. Propolis is a natural plant product; it acts as a powerful reactive oxygen species scavenger and improves the neurodegeneration process. In this study 40 adult male albino rats were divided randomly into four groups 10 rats each: group I (control group), group II manganese chloride (MnCl2) received 10 mg/kg/day/orally for 4 weeks by intra-gastric tube, group III (propolis group) received 50 mg/kg/day/orally for 4 weeks by intra-gastric tube, and group IV (MnCl2 + propolis group) received the same doses with the same duration and route as in groups II and III. Rats were sacrificed after 24 h of last dose. The olfactory bulbs removed, the right bulb cut to be processed for haematoxylin and eosin, immunohistochemical staining and the left cut for electron microscopic studies. Results revealed that rat olfactory bulb from MnCl2 group showed darkly stained mitral cells with dark pyknotic nuclei, some show pericellular spaces and vacuolation, dark apoptotic cells in granular cells, neuropil vacuolation and pyknotic astrocyte. Electron microscopic examination showed abnormal granular cell with irregular damaged nuclear membrane, rupture of myelin fibre. Mitral nerve cell with destructed nucleus, many cytoplasmic vacuoles, swollen rough endoplasmic reticulum, vacuolated mitochondria and neuropil were observed. Manganese chloride + propolis group showed improvement compared to MnCl2 group. It was concluded that propolis can ameliorate the toxic changes of manganese chloride on rat olfactory bulb.

DOES ORAL CHLOROQUINE AFFECT THE HIPPOCAMPUS IN RATS? A CLUE TO CHLOROQUINE INDUCED NEUROPSYCHIATRIC ADVERSE EFFECTS

Background: Chloroquine is widely used in medicine. The main indication for its usage is treatment of malaria. Reportsabout psychiatric side effects of chloroquine are rare. However, the list of recorded chloroquine induced neuropsychiatricdisorders shows wide range of symptoms. Aim of the work: In this study, the effect of oral chloroquine on the hippocampus of rats was assessed. Material and methods: Twenty seven male adult albino rats were randomly divided into four groups. Group I (control group): nine rats were divided into I-a and I-b subgroups. Group II (chloroquine for two weeks): six rats received 4ml of distilled water solution/day containing chloroquine in a dose of 80 mg/kg bwvia oral gavage for two weeks. Group III (chloroquine for three weeks): six rats received chloroquine as in group II for three weeks. Group IV (chloroquine for four weeks): six rats received chloroquine as in group II for four weeks. After sacrifice, the hippocampi were retrieved, fixed, and processed for paraffin sections. H&E and Bielschowsky silver stains were applied and immunehistochemical staining for GFAP was performed to examine the distribution of astrocytes. Results: Examination of different regions of the hippocampal formation revealed dark, shrunken cells, with pyknotic nuclei and pericellular spaces in all treated groups. Neurofibrillary tangles were also seen in some stained sections. Moreover, an increase in the density of astrocytes was also observed. Morphometrically, there was a decrease in the thickness of both pyramidal and granular layers of cornu ammonis and dentate gyrus respectively in all treated groups as compared with control group. All these changes appeared in group II, and were clearer in both groups III and IV. Conclusion: It was concluded that oral administration of chloroquine caused duration dependent neuronal damage in the hippocampus of rats giving a possible explanation for chloroquine induced neuropsychiatric adverse effects.

Apoptotic Osteocytes Induce RANKL Production in Bystanders via Purinergic Signaling and Activation of Pannexin Channels.

Localized apoptosis of osteocytes, the tissue-resident cells within bone, occurs with fatigue microdamage and activates bone resorption. Osteoclasts appear to target and remove dying osteocytes, resorbing damaged bone matrix as well. Osteocyte apoptosis similarly activates bone resorption with estrogen loss and in disuse. Apoptotic osteocytes trigger viable neighbor (ie, bystander) osteocytes to produce RANKL, the cytokine required for osteoclast activation. Signals from apoptotic osteocytes that trigger this bystander RANKL expression remain obscure. Studying signaling among osteocytes has been hampered by lack of in vitro systems that model the limited communication among osteocytes in vivo (ie, via gap junctions on cell processes and/or paracrine signals through thin pericellular fluid spaces around osteocytes). Here, we used a novel multiscale fluidic device (the Macro-micro-nano, or Mμn) that reproduces these key anatomical features. Osteocytes in discrete compartments of the device communicate only via these limited pathways, which allows assessment of their roles in triggering osteocytes RANKL expression. Apoptosis of MLOY-4 osteocytes in the Mμn device caused increased osteocyte RANKL expression in the neighboring compartment, consistent with in vivo findings. This RANKL upregulation in bystander osteocytes was prevented by blocking Pannexin 1 channels as well as its ATP receptor. ATP alone caused comparable RANKL upregulation in bystander osteocytes. Finally, blocking Connexin 43 gap junctions did not abolish osteocyte RANKL upregulation, but did alter the distribution of RANKL expressing bystander osteocytes. These findings point to extracellular ATP, released from apoptotic osteocytes via Panx1 channels, as a major signal for triggering bystander osteocyte RANKL expression and activating bone remodeling. © 2020 American Society for Bone and Mineral Research.

The place of neurotrophic therapy in the concept of stimulation of secondary angiogenesis in acute focal cerebral ischemia

The features of changes in the structural and functional characteristics of brain tissue were studied in 60 outbred male Wistar rats during modelling of acute focal cerebral ischemia (AFCI) in the dynamics of treatment with human cryopreserved cord blood serum (CCBS). Electronic and optical microscopic examination of the sensorimotor area of the cerebral cortex was performed. All animals were divided into three groups: the first (control) group consisted of intact rats without trauma and treatment; the second group consisted of animals after modelling AFCI without treatment; third group consisted of rats after modelling AFCI, which were injected by CCBS. Each group consisted of 20 animals. Material for morphological examination was taken after administration of CCBS solution to animals with the model of AFCI at 12, 24, 72 hours and 7th days after the start of the experiment. The average area of perivascular spaces, which is an indicator of vasogenic oedema in rats of group 2 was 45 times higher than in rats of group 1. In contrast, in rats of group 3 with CCBS, this figure was exceeded 37 times. The average area of pericellular spaces, indicating the degree of cytotoxic oedema, in animals of group 2 on the 7th day after AFCI was almost 23 times higher than the results of group 1. This figure in rats of group 3 was increased 20 times compared with data in group 2. Against the background of the use of this drug, there were signs of reactive changes in endothelial cells in the form of an increase in the number of ribosomes and polysomes in the cytoplasm, a decrease in the degree of perivascular oedema of brain tissue by 21.4 %. The surface area of endothelial cells in the zone of AFCI on the 7th day of the experiment in animals that additionally received CCBS was (1483.00±26.48) μm2, which indicates a positive anti-inflammatory effect of the drug. On the 7th day of the experiment in group 3 rats by optical microscopy was found to increase the density of cerebral capillaries compared with group 2, which indicates the stimulation of the restoration of the ultrastructure of damaged capillaries, increase their density, the formation of new capillaries under the components of CCBS.

Effect of Energy Drinks on Rat Hippocampus and the Possible Neuroprotective Role of Sesame Oil: Histological, Immunohistochemical and Molecular Study

AbstractBackground: Energy drinks (EDs) consumption has in-creased dramatically over the past years and several adverse effects have been reported. Sesame oil (SO) is a potent antioxidant and anti-inflammatory dietary source for human health.Aim of Study: The present study was assigned to investigate the possible neuroptotecive role of SO on energy drinks-induced hippocampal damage.Material and Methods: Twenty adult male albino rats were divided into four groups; five rats in each (control, SO, EDs and EDs+SO). Routine histological examination was done, together with toluidine blue stain for dark neurons (DN) and glial fibrillary acidic protein (GFAP) immunostaining for astrocytes. Gene expression of catalase and tumor necrosis factor alpha (TNF-a) was analyzed using real-time polymerase chain reaction (PCR) and histomorphometric analysis was done.Results: Histological examination of the hippocampus from EDs group showed shrunken pyramidal and granule cells, vacuolation and large pericellular spaces. There was significant decrease in neuronal thickness, increase in the number of DN and GFAP area percent compared to control group. Significant decrease in catalase gene expression was observed along with increase in TNF-a gene expression. Concomitant SO administration significantly improved the previously mentioned changes.Conclusion: Energy drinks administration induced mor-phological and morphometric changes, deficient antioxidant capacity and inflammatory response in rat hippocampus. Sesame oil administration alleviated these adverse changes possibly due to its antioxidant and anti-inflammatory prop-erties.

THE PERSPECTIVES OF CELLULAR THERAPY IN REHABILITATION TREATMENT OF NEUROLOGICAL PATIENTS (REVIEW OF LITERATURE WITH OWN RESEARCH DATA)

The article gathers and analyses modern data from foreign and regional authors regarding the latest achievements in cellular technologies that have prospects in rehabilitation of neurological patients. For the replacement of damaged brain tissue, technologies directed for differentiation of stem cells in neuronal and glial directions are extremely promising. Today, it is feasible to expect that in the near future this will enable the transplantation of cells to activate the processes of neuroplasticity in recovery period. The data of own researches, which examined changes in structural and functional characteristics of brain tissue of rats with condition of experimental acute focal cerebral ischemia (AFCI) in dynamics of treatment cryopreserved cord blood serum (CCBS) was given for evaluation of its membrane protection, immune modulation and proangiogenic activity. The study was conducted on 60 outbred white male Wistar rats weighing 200±20 g. All animals were divided into 3 groups: 1st group (controls) – intact rats without trauma and treatment; 2nd group – animals after modelling AFCI without treatment; 3rd group – rats after modelling AFCI, which was administered CCBS. The results of the study indicated the stimulating effect of the components of CCBS on restoration of ultrastructure of the damaged capillaries, increasing their density, as well as the formation of new capillaries. It was found that the average area of the perivascular spaces, which is an indicator of vasogenic edema in rats of group 2 is 45 times higher than that in group 1, while in rats in group 3 treated with CCBS, this figure was exceeded 37 times. The average area of pericellular spaces, indicating the degree of cytotoxic edema, in rats of group 2 on the 7th day after AFCI is almost 23 times higher than the results of group 1. This indicator in rats of group 3 was increased by 20 times compared with group 2. On the 7th day of the experiment in rats of group 2, the surface area of endothelial cells was significantly larger than in rats of groups 1 and 3 by 54.1% and 31.6% respectively. Neurotrophic therapy is a very promising area of regenerative medicine, which requires further study in the use of growth factors.

The morphological characteristic of the cerebellar cortex in the case of a burning injury

The objective of the present study was the development of the additional forensic medical criteria for the diagnostics of the intravitality and prescription of the burning injury making use of the morphological changes in the cerebellar cortex. A total of 82 cases of the death from the second- and third a, b-degree flame burns in 63 men and 19 women at the age from 20 to 65 years were available for the analysis. The condition of the cerebellar cortex was evaluated within 0 to 72 hours after the trauma had been inflicted. The routine histological staining technique using hematoxylin and eosin were employed as well as the Nisslin staining carried out in the combination with the immunohistochemical reaction based on the application of the antibodies against the glial fibrillary acidic protein (GFAP). In the case of death during the acute period after the burning injury, the histological study revealed a characteristic complex of the morphological features including the acute swelling of neurons, the increasing expansion of perivascular and pericellular spaces, as swell as hyperoxyphilia of microglia. The astrocytes of cortical II-III layers proved highly sensitive to tissue hypoxia as appears from their reaction with anti-GFAP antibodies. It is concluded that the results of the evaluation of the blood supply of the cerebral hemisphere cortex with the use of immunohistochemical methods may be helpful as the additional criteria for the purpose of forensic medical documentation of intravitality and prescription of the burning injury.

125 Characterization of TSG-6 protein in healthy and inflammatory models of reconstructed human epidermis

TSG-6 is an anti-inflammatory hyaluronan-binding protein involved in extracellular matrix remodelling whose functions have never been studied in skin. Characterization of TSG-6 was carried out in vivo in healthy human epidermis and in vitro using keratinocyte monolayers and reconstructed human epidermis (RHE). Distribution of TSG-6 protein was highlighted by immunofluorescence and levels of relative mRNA and protein expression were respectively measured by RT-qPCR and ELISA. The immunofluorescent signal of TSG-6 protein was located in the living layers of epidermis in vivo and in vitro, mostly in basal keratinocytes, and was restricted to the intracellular compartment. Because the localization of hyaluronan (HA) is in the extracellular matrix, no colocalization of TSG-6 and HA could thus be detected. ELISA assay shows that TSG-6 is abundant in the intracellular compartment, and is present in weak amounts in pericellular spaces, as well as in the culture medium. In vitro, TSG-6 mRNA level did not change during epidermal differentiation induced by confluence and increased cell density. The expression of TSG-6 was then analysed in RHE subjected to inflammatory conditions using two different models. In the first one, RHE were infected with arthrospores of Trichophyton rubrum dermatophyte, allowing adhesion and invasion of cornified layer by fungal elements. The second one corresponds to an atopic dermatitis-like model where RHE were first deprived from plasma membrane cholesterol using methyl-b-cyclodextrin ahead of exposure to interleukins (IL-4, IL-13, IL-25). In both inflammatory models, despite TSG-6 mRNA and protein levels were increased, simultaneously with an increase in HA fluorescent detection no colocalization of TSG-6 with HA was ever detected. In summary, these results demonstrate that TSG-6 is present intracellularly in epidermal keratinocytes in physiological conditions, and its expression is induced by inflammatory conditions. Only low concentration of this protein is detectable extracellularly.

A characteristic chondroitin sulfate trisaccharide unit with a sulfated fucose branch exhibits neurite outgrowth-promoting activity: Novel biological roles of fucosylated chondroitin sulfates isolated from the sea cucumber Apostichopus japonicus

Chondroitin sulfate (CS) is a class of sulfated glycosaminoglycan (GAG) chains that consist of repeating disaccharide unit composed of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc). CS chains are found throughout the pericellular and extracellular spaces and contribute to the formation of functional microenvironments for numerous biological events. However, their structure-function relations remain to be fully characterized. Here, a fucosylated CS (FCS) was isolated from the body wall of the sea cucumber Apostichopus japonicus. Its promotional effects on neurite outgrowth were assessed by using isolated polysaccharides and the chemically synthesized FCS trisaccharide β-D-GalNAc(4,6-O-disulfate) (1–4)[α-l-fucose (2,4-O-disulfate) (1–3)]-β-D-GlcA. FCS polysaccharides contained the E-type disaccharide unit GlcA-GalNAc(4,6-O-disulfate) as a CS major backbone structure and carried distinct sulfated fucose branches. Despite their relatively lower abundance of E unit, FCS polysaccharides exhibited neurite outgrowth-promoting activity comparable to squid cartilage-derived CS-E polysaccharides, which are characterized by their predominant E units, suggesting potential roles of the fucose branch in neurite outgrowth. Indeed, the chemically synthesized FCS trisaccharide was as effective as CS-E tetrasaccharide in stimulating neurite elongation in vitro. In conclusion, FCS trisaccharide units with 2,4-O-disulfated fucose branches may provide new insights into understanding the structure-function relations of CS chains.

Roles and Modalities of Ectonucleotidases in Remodeling the Multiple Myeloma Niche.

Ectoenzymes are cell surface molecules, which represent functional bridges between the environment and the cytoplasm. One set of ectoenzymes—CD39, CD38, CD203a, and CD73—leads to the generation of adenosine (ADO) by metabolizing ATP and NAD+. While ADO is known to control inflammation and suppress immune responses, other aspects of ADO function are still obscure, mainly due to its short half-life in biological fluids. Human multiple myeloma (MM) grows in the closed system of the bone marrow (BM) niche representing an ideal setting for studying ectoenzymes and their products. Another source of information on ectoenzyme function may derive from in vivo results of anti-CD38 antibody therapy in MM. Current results, obtained from in vitro models and from preliminary in vivo findings, indicate that ectoenzymes produce ADO locally in the BM niche. Furthermore, MM cells release microvesicles (MV), which thanks to their molecular cargo and surface ectoenzymes may function as particulate communicators outside of the niche. During anti-CD38 antibody therapy, the MV carry therapeutic IgG, determining that the prevalent orientation of MV will be toward cells and tissues expressing receptors for the IgG Fc domain. The resulting picture is one where MM adopts an immune escape strategy based on reshaping the environmental niche. This adaptation is followed by actions of MV that are exerted in biological fluids and circulating immune cells. By coating FcRs+ cells, MV modify pericellular spaces, reproducing the metabolic halo generated by ectoenzymes within closed systems.

Colonization and Intrusive Invasion of Potato Psyllid by 'Candidatus Liberibacter solanacearum'.

Previous studies have shown that the fastidious bacterial plant pathogen 'Candidatus Liberibacter solanacearum' (CLso) is transmitted circulatively and propagatively by the potato psyllid (PoP) Bactericera cockerelli. In this study, the temporal and spatial interrelationships between CLso PoP were investigated by scanning electron microscopy of the digestive system of PoP immature and adult instars and salivary glands of adults post CLso ingestion. CLso biofilms were not detectable on the outer midgut surface of the first and second instars; however, for third to fifth instars and teneral and mature adults, biofilms were observed in increasing numbers in each successive developmental stage. In adult PoP midguts, CLso cells were observed between the basal lamina and basal epithelial cell membranes; in basal laminar perforations, on the outer basal laminar surface, and in the ventricular lumen, epithelial cytosol, and filter chamber periventricular space. CLso were also abundantly visible in the salivary gland pericellular spaces and in the epidermal cell cytosol of the head. Collectively, these results point to an intrusive, systemic invasion of PoP by CLso that employs an endo/exocytosis-like mechanism, in the context of a propagative, circulative mode of transmission.